Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (18S, 28S, EF, UBA, UBQ, TIF, NTB, TUA, RPS, and TIF5A) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S, TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

The WRKY family of transcription factors (TFs) includes a number of transcription-specific groupings that play important roles in plant growth and development and in plant responses to various stresses. To screen for WRKY transcription factors associated with drought stress in Zanthoxylum bungeanum, a total of 38 ZbWRKY were identified and these were then classified and identified with Arabidopsis WRKY. Using bioinformatics analyses based on the structural characteristics of the conservative domain, 38 WRKY transcription factors were identified and categorized into three groups: Groups I, II, and III. Of these, Group II can be divided into four subgroups: subgroups IIb, IIc, IId, and IIe. No ZbWRKY members of subgroup IIa were found in the sequencing data. In addition, 38 ZbWRKY were identified by real-time PCR to determine the behavior of this family of genes under drought stress. Twelve ZbWRKY transcription factors were found to be significantly upregulated under drought stress and these were identified by relative quantification. As predicted by the STRING website, the results show that the WRKYs are involved in four signaling pathways—the jasmonic acid (JA), the salicylic acid (SA), the mitogen-activated protein kinase (MAPK), and the ethylene signaling pathways. ZbWRKY33 is the most intense transcription factor in response to drought stress. We predict that WRKY33 binds directly to the ethylene synthesis precursor gene ACS6, to promote ethylene synthesis. Ethylene then binds to the ethylene activator release signal to activate a series of downstream genes for cold stress and osmotic responses. The roles of ZbWRKY transcription factors in drought stress rely on a regulatory network center on the JA signaling pathway.

Zanthoxylum bungeanum Maxim. (Z. bungeanum), a plant that belongs to the Rutaceae family, is widely planted in China. Its outstanding feature is its rich aroma. The main component that creates this aroma is the volatile terpenoids. In this study, we aimed to understand the molecular mechanism related to aroma synthesis in Z. bungeanum and provide new ideas for breeding. Headspace solid phase micro extraction-gas chromatography mass spectrometry (HS-SPME-GC-MS), RT-qPCR and bioinformatics were used to study the changes in volatile terpenoids and identify key genes in the pathway of terpenoids in fruits of Z. bungeanum. The results show that the trend of volatile terpenoids is consistent among the two varieties. As the fruit matures, the terpenoids gradually accumulate and peak in the third period (mid-development) before gradually decreasing. Among these terpenoids, there is the highest content of α-pinene. In Z. bungeanum cv. ‘Hanchengdahongpao’ (Hanchengdahongpao) and Z. bungeanum cv. ‘Fuguhuajiao’ (Fuguhuajiao), this reached 24.74% and 20.78% respectively. In general, for the content of volatile terpenoids, Hanchengdahongpao is 62% and Fuguhuajiao is 41%. The results of RT-qPCR showed that most gene expression in this study was upregulated. Among them, ZbDXS has the highest relative level of expression in itself, which is the key rate-limiting enzymein the MEP pathway. These results explore the synthetic route of terpenoids during the ripening process of Z. bungeanum, which provides a reference for cultivar and improving good traits.

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the largest plant-specific TF families and play a pivotal role in adaptation to abiotic stresses. The genome-wide analysis of NAC TFs is still absent in Zanthoxylum bungeanum. Here, 109 ZbNAC proteins were identified from the Z. bungeanum genome and were classified into four groups with Arabidopsis NAC proteins. The 109 ZbNAC genes were unevenly distributed on 46 chromosomes and included 4 tandem duplication events and 17 segmental duplication events. Synteny analysis of six species pairs revealed the closely phylogenetic relationship between Z. bungeanum and C. sinensis. Twenty-four types of cis-elements were identified in the ZbNAC promoters and were classified into three types: abiotic stress, plant growth and development, and response to phytohormones. Co-expression network analysis of the ZbNACs revealed 10 hub genes, and their expression levels were validated by real-time quantitative polymerase chain reaction (qRT-PCR). Finally, ZbNAC007, ZbNAC018, ZbNAC047, ZbNAC072, and ZbNAC079 were considered the pivotal NAC genes for drought tolerance in Z. bungeanum. This study represented the first genome-wide analysis of the NAC family in Z. bungeanum, improving our understanding of NAC proteins and providing useful information for molecular breeding of Z. bungeanum.

When under drought, plants activate a range of self-protective responses. Among these are activation of the antioxidant system and changes in stomatal aperture. The antioxidant system can remove the reactive oxygen species produced under drought conditions and so mitigate oxidative damage. Water becomes a severely limiting resource for plants suffering drought stress, so they generally close their stomata to reduce water loss. We examined changes in the activities of the antioxidant enzymes and altered gene expression patterns in Zanthoxylum bungeanum plants exposed to drought by irrigation with 20% PEG6000. We also recorded changes in stomatal aperture as the drought persisted. Relationships between the antioxidant system and stomatal aperture were analyzed in relation to gene expression. The results indicate that under drought stress, POD, CAT, APX, proline, MDA and related genes all show positive responses to drought, while SOD and its genes showed a negative response. The relationship between drought duration and stomatal aperture was considered. Stomatal aperture declines exponentially as drought duration increases.